The NM_000152.5:c.953T>C variant in GAA is a missense variant predicted to cause substitution of methionine by threonine at amino acid 318 (p.Met318Thr). Twelve probands with a diagnosis of Pompe disease were identified with this variant. At least 9 of them have GAA activity in the affected range in dried blood spots or <30% normal GAA activity in fibroblasts (PMIDs: 1652892, 19862843, 25139343, 30214072, 34357340, clinical laboratory data) with pseudodeficiency variants confirmed absent in three of these patients (clinical laboratory data) (PP4_Moderate). Of these patients, 10 are compound heterozygous for c.953T>C (p.Met318Thr) and a variant in GAA that has been classified as pathogenic by the ClinGen LSD VCEP; either c.2560C>T (p.Arg854Ter), confirmed in trans (PMID: 1652892, 19862843), c.1979G>A (p.Arg660His), confirmed in trans by parental testing (clinical diagnostic laboratory); c.-32-13T>G, phase unknown (PMIDs: 29122469, 31904026, 32317649, clinical diagnostic laboratory) (at least 5 patients, 1 confirmed in trans) c.2481+102_2646+31del (p.Gly828_Asn882del), phase unknown (clinical diagnostic laboratory), c.1292_1295dupTGCA, phase unknown (PMID: 30214072), or c.1082C>T (p.Pro361Leu), phase unknown (PMID: 25139343). One patient is homozygous for the variant (PMID: 34357340) (0.5 points) (PM3_Very Strong). Another patient is compound heterozygous for the variant and c.692+5G>T (PMID: 29181627) but the allelic data from this patient will be used in the classification of c.692+5G>T and is not included here to avoid circular logic. The highest population minor allele frequency in gnomAD v2.1.1 is 0.00009 in the African population, which is lower than the ClinGen LSD VCEP threshold (<0.001) for PM2_Suporting, and therefore meets this criterion (PM2_Supporting). Functional studies involving expression of the variant in cultured cells indicate that the variant impacts function, resulting in <2% GAA activity (PMID: 1652892, 19862843). The computational predictor REVEL gives a score of 0.89, which is above the threshold of 0.7, evidence that correlates with impact to GAA function (PP3). There is a ClinVar entry for this variant (Variation ID: 4021; 1 star review status) with 3 submitters classifying the variant as pathogenic, two as likley pathogenic and one as a variant of uncertain significance. With data from the published literature and a clinical laboratory, we find that the variant meets the criteria to be classified as pathogenic for Pompe disease. GAA-specific ACMG-AMP criteria met, as specified by the ClinGen LSD VCEP (Specifications Version 2.0): PM3_Very Strong, PP4_Moderate, PS3_Supporting, PM2_Supporting, PP3. Classification approved by the ClinGen LSD VCEP on Sept. 6, 2022.

A heterozygous missense variant was identified, NM_000152.3(GAA):c.953T>C in exon 5 of 20 of the GAA gene. This substitution is predicted to create a moderate amino acid change from methionine to threonine at position 318 of the protein, NP_000143.2(GAA):p.(Met318Thr). The methionine at this position has moderate conservation (100 vertebrates, UCSC), and is located in the N-terminal of the glycosyl-hydrolase domain of the glycoside hydrolase family 31 (GH31). In silico software predicts this variant to be disease causing (Polyphen, SIFT, CADD, Mutation Taster). The variant is present in the gnomAD population database at a frequency of 0.005% (10 heterozygotes, 0 homozygotes). The variant has been previously reported as pathogenic multiple times (ClinVar, Zhong N. et al (1991), Kroos M., et al (2008) and Bali D.S. et al (2011)). The POMPE database lists the variant as CRIM positive and potentially less severe (Kroos M., et al (2008) and Bali D.S. et al (2011)). Functional studies show that this variant causes enzyme deficiency with residual activity (Bali D.S. et al (2011)). Based on information available at the time of curation, this variant has been classified as PATHOGENIC.

The heterozygous p.Met318Thr variant in GAA has been reported in 2 individuals with Glycogen Storage Disease II (PMID: 1652892, 25139343), and has also been reported pathogenic by OMIM, likely pathogenic by Integrated Genetics, and a VUS by EGL Genetic Diagnostics in ClinVar (Variation ID: 4021). This variant has been identified in 0.009% (2/23234) of African chromosomes and 0.006% (7/121686) of European (non-Finnish) chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP rs121907936). Although this variant has been seen in the general population, its frequency is low enough to be consistent with a recessive carrier frequency. In vitro functional studies with a minigene assay provide some evidence that the p.Met318Thr variant may eliminate GAA activity (PMID: 1652892). However, these types of assays may not accurately represent biological function. Computational prediction tools and conservation analyses suggest that this variant may impact the protein, though this information is not predictive enough to determine pathogenicity. However, the Methionine (Met) at position 318 is not conserved in evolutionary distant species, raising the possibility that a change at this position may be tolerated. Two additional variants at the same position, p.Met318Lys and p.Met318Leu, have been reported as VUS in ClinVar but p.Met318Lys is a likely pathogenic variant, slightly increasing the likelihood that the p.Met318Thr variant is pathogenic (Variation ID: 558700, 290616; PMID: 21484825, 22644586). This variant has been reported in combination with a reported likely pathogenic variant and a variant reported to cause NMD, and in individuals with Glycogen Storage Disease II (PMID: 1652892, 25139343). In summary, although additional studies are required to fully establish its clinical significance, this variant is likely pathogenic. ACMG/AMP Criteria applied: PS3, PM2, PM5_Supporting, PP3, PP4 (Richards 2015).

Variant summary: GAA c.953T>C (p.Met318Thr) results in a non-conservative amino acid change located in the Glycoside hydrolase family 31, N-terminal domain (IPR025887) of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 4.2e-05 in 236242 control chromosomes. This frequency is not significantly higher than expected for a pathogenic variant in GAA causing Glycogen Storage Disease, Type 2 (Pompe Disease) (4.2e-05 vs 0.0042), allowing no conclusion about variant significance. c.953T>C has been reported in the literature in several individuals affected with Glycogen Storage Disease, Type 2 (Pompe Disease) (ie. Zhong_1991, Flanagan_2009, Mori_2017, Loscher_2018, Kazi_2019). These data indicate that the variant is likely to be associated with disease. Publications also reported experimental evidence evaluating an impact on protein function, and demonstrated that the variant results in <10% of normal activity (Zhong_1991, Flanagan_2009). Seven clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. Six labs classified as likely pathogenic/pathogenic while one classified as VUS. Based on the evidence outlined above, the variant was classified as pathogenic.

Not observed at significant frequency in large population cohorts (gnomAD); Published functional studies demonstrate a damaging effect with <2% residual GAA enzyme activity (Zhong et al., 1991; Flanagan et al., 2009); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 19862843, 21228398, 29181627, 30214072, 31254424, 22253258, 19343043, 34357340, 25139343, 31342611, 31086307, 29122469, 30155607, 31904026, 32317649, 34576242, 1652892)

The c.953T>C variant in GAA is a missense variant predicted to cause substitution of methionine to threonine at amino acid 318. This variant is rare in the general population with a frequency below the threshold expected for the associated phenotype(s). This variant has been observed in one or more individuals affected with the associated recessive disease, as either homozygous or compound heterozygous with a second variant (PMID: 29122469, 32317649, 30155607). Given the available evidence, this variant is classified as Pathogenic.

Positive for the pathogenic variant c.953T>C (p.M318T) in the GAA gene. If this individual's partner is a carrier for GLYCOGEN STORAGE DISEASE, TYPE 2 (POMPE DISEASE), their chance to have a child with this condition is 1 in 4 (25%). Carrier screening for this individual's partner is suggested. The c.953T>C (p.M318T) variant in the GAA gene has been observed at a frequency of 0.0045% in the gnomAD v2.1.1 dataset. This variant has been reported in a homozygous state or in conjunction with another variant in individual(s) with glycogen storage disease, type 2 (Pompe disease) (PMID: 1652892, 21228398, 29181627, 29122469). Functional studies demonstrated that this variant causes reduced enzyme activity (PMID: 1652892).

This sequence change replaces methionine, which is neutral and non-polar, with threonine, which is neutral and polar, at codon 318 of the GAA protein (p.Met318Thr). This variant is present in population databases (rs121907936, gnomAD 0.009%). This missense change has been observed in individuals with Pompe disease (PMID: 1652892, 19862843, 29122469, 29181627). ClinVar contains an entry for this variant (Variation ID: 4021). An algorithm developed to predict the effect of missense changes on protein structure and function (PolyPhen-2) suggests that this variant is likely to be disruptive. Experimental studies have shown that this missense change affects GAA function (PMID: 1652892, 19862843). This variant disrupts the p.Met318 amino acid residue in GAA. Other variant(s) that disrupt this residue have been observed in individuals with GAA-related conditions (PMID: 21484825), which suggests that this may be a clinically significant amino acid residue. For these reasons, this variant has been classified as Pathogenic.

GAA p.Met318Thr (c.953T>C) is a missense variant that changes the amino acid at codon 318 from Methionine to Threonine. This variant has been observed in at least one proband with a GAA-related disorder in the compound heterozygous and/or homozygous state (PMID:19862843;30214072;29181627;34357340;31904026;25139343). At least one functional study has demonstrated a substantial alteration in protein function relative to the wild-type (PMID:19862843;1652892). It is absent or not present at a significant frequency in gnomAD. In silico models predict that this variant is possibly or probably damaging. In conclusion, we classify GAA p.Met318Thr (c.953T>C) as a likely pathogenic variant.